The human complement system constitutes an important part of innate immunity, which has three major physiological activities (Walport, “Complement. First of two parts,” New Engl. J. Med. 344:1058-1066 (2001) Walport, “Complement. Second of two parts,” New Engl. J. Med. 344:1140-1144 (2001)). First, it defends the host against bacterial infections through opsonization (a process that makes antigens more susceptible to phagocytosis), activation of leukocytes, and lysis of bacterial cells. Second, it connects innate and acquired immunity by augmenting antibody response and enhancing immunologic memory. Third, it disposes of waste from tissue such as immune complexes, apoptotic cells, and products of inflammatory injury. The complement system has about 35 cell surface and soluble plasma proteins. In plasma, they amount to more than 3 g per liter and constitute approximately 15% of the globulin fraction. These proteins are synthesized mainly in the liver.
The complement system is activated through three pathways, all of them leading to the generation of the homologous variants of the protease C3-convertase (Walport, “Complement. First of two parts,” New Engl. J. Med. 344:1058-1066 (2001); Walport, “Complement. Second of two parts,” New Engl. J. Med. 344:1140-1144 (2001)). As shown in FIG. 1, the classical complement pathway typically requires antibodies for initiation, while the alternate pathway can be activated by spontaneous C3 hydrolysis (“tickover”) without the presence of antibodies. The mannose-binding lectin pathway belongs to the non-specific immune response as well. C3-convertase cleaves and activates component C3, creating C3a and C3b and causing a cascade of further cleavage and activation events. C3b binding to the surface of pathogens (opsonization) leads to recognition by C3 receptors, such as CR1 or CR3 on phagocytic cells, which enhances the engulfment of the opsonized particles. C5b initiates the membrane attack pathway, which results in formation of the membrane attack complex (“MAC”), including C5b, C6, C7, C8, and polymeric C9. In addition to these two major effector mechanisms (opsonization and the formation of the MAC), there are other effects of complement activation. For example, C5a is an important chemotactic factor that helps recruit inflammatory cells. Both C3a and C5a have anaphylatoxin activity, which results in mast cell degranulation, increased vascular permeability, smooth muscle contraction, etc.
The complement system emerged about 600-700 million years ago and over the course of evolution it has been endowed with a wide range of functions. Unlike components involved in acquired immunity, complement proteins do not have the intrinsic capability to discriminate between self and non-self antigens, and have the potential to damage or destroy any particles (molecules, supramolecular assemblies, or cells) to which they bind. Therefore, in addition to its role in immune defense, the complement system contributes to tissue damage in many clinical conditions. As a means of preventing homologous attack, the complement system is tightly regulated by a group of proteins called regulators of complement activation (“RCA”), and host cell damage is often the consequence of unregulated complement activation. While complement activation is not an etiological factor in any known disease, it may play an essential role in the pathogenesis of many diseases. Tissue injury can be caused directly by MAC or indirectly by the generation of the anaphylatoxic peptides C3a and C5a.
Complement has been implicated in numerous infectious, allergic, biocompatibility, shock, rheumatological, renal, hematological, dermatological, neurological, and vascular/pulmonary diseases and disorders. Therefore, there is a pressing need for effective complement inhibitors to prevent and treat these disease states (Sahu et al., “Complement Inhibitors: A Resurgent Concept in Anti-Inflammatory Therapeutics,” Immunopharmacology 49:133-148 (2000)). Ideally, the choice of complement protein or proteins as drug targets should be made according to the specific pathological condition of interest. Following this logic, complement inhibitors have been developed for a few targets. However, in most clinical conditions, it is difficult to determine which pathway initiated the activation, and activation of one pathway often leads to the recruitment of another. Therefore, a complement inhibitor that blocks all three pathways would be useful. Because all three pathways converge at the C3 activation step, blocking this step would inhibit C3a and C5a generation and MAC formation, which are implicated in complement-mediated damage of host cells.
There are several types of C3 inhibitors being developed. One type of inhibitor makes use of naturally occurring proteins that bind to C3 or its derivatives, in particular cell surface complement receptors. The recombinant form of these receptors lacking the transmembrane region and the cytoplasmic tail of the parent molecule is soluble and able to neutralize C3 through binding. The first recombinant complement inhibitor was soluble CR1 (“sCR1”), which has been tested in clinical trials for complement inhibition in ischemia-reperfusion injury (Weisman et al., “Soluble Human Complement Receptor Type 1: In Vivo Inhibitor of Complement Suppressing Post-ischemic Myocardial Inflammation and Necrosis,” Science 249:146-151 (1990)). Recently, the extracellular domain of human complement receptor of the immunoglobulin superfamily (“CRIg”) has also been developed to serve as an inhibitor of the alternative pathway and shown to reverse inflammation and bone destruction in experimental arthritis (Katschke et al., “A Novel Inhibitor of the Alternative Pathway of Complement Reverses Inflammation and Bone Destruction in Experimental Arthritis” The Journal of Experimental Medicine 204:1319-1325 (2007)). Another type of C3 inhibitor is a peptide aptamer such as Compstatin, a 13-residue cyclic peptide isolated from a combinatorial library (Sahu et al., “Inhibition of Human Complement by a C3-binding Peptide Isolated from a Phage-displayed Random Peptide Library,” Journal of Immunology 157:884-891 (1996)). This peptide binds to C3 and inhibits its cleavage by C3 convertase. Its effect has been tested in several clinically relevant models.
Aptamers have previously been designed for uptake by a specialized structure called the flagellar pocket of the parasitic organism Trypanosoma brucei, with the goal of directing RNA-conjugated toxins to the lysosomal compartment of the organism as a therapy to treat parasitic infection in humans (Homann et al., “Uptake and Intracellular Transport of RNA Aptamers in African Trypanosomes Suggest Therapeutic ‘Piggy-back’ Approach,” Bioorg. Med. Chem. 9:2571-2580 (2001)). However, this approach was limited to directing a toxin-conjugated aptamer to a specific compartment within a parasitic organism and not designed to exploit a receptor on the organism's surface or make use of the lysosome's function to break down aptamer-bound endogenous or exogenous molecules. Toxins considered for this approach, with the intention of disrupting lysosomes using molecules that undergo conformational changes on encountering a pH shift, included poly(2-ethyl acrylic acid), poly(lysine dodecanamide), and melittin, a component of honeybee venom (Goringer et al., “RNA Aptamers as Potential Pharmaceuticals Against Infections with African Trypanosomes,” Handbook of Experimental Pharmacology 173:375-93 (2006)). It has also been suggested that targeting the trypanosome's surface with aptamers conjugated to antigens renders the parasites susceptible to recognition by antibodies (Lorger et al., “Targeting the Variable Surface of African Trypanosomes with Variant Surface Glycoprotein-specific, Serum-stable RNA Aptamers,” Eukaryotic Cell 2(1):84-94 (2003)). More recently, a nanobody targeting strategy has been developed (“Experimental Therapy of African Trypanosomiasis with a Nanobody-conjugated Human Trypanolytic Factor,” Nature Medicine 12(5):580-4 (2006)). However, none of these approaches involved promoting destruction of endogenous or exogenous molecules by targeting these molecules to the lysosome.
The idea of conscripting the complement system, especially the alternative pathway, in cancer immunotherapy was proposed more than 20 years ago, but then neglected for a long time as the major emphasis was put on cell-mediated immune response against cancer (Cooper, “Complement and Cancer: Activation of the Alternative Pathway as a Theoretical Base for Immunotherapy,” Advances in Immunity and Cancer Therapy 1:125-166 (1985)). However, with the introduction of monoclonal antibodies (“mAbs”), complement has come into play with great potential as an effector system in cancer immunotherapy (Macor et al., “Complement as Effector System in Cancer Immunotherapy,” Immunology Letters 111:6-13 (2007)). Most mAbs that mediate antibody-dependent cellular cytotoxicity (“ADCC”) also activate the complement system. Complement has a number of advantages over other systems in that it is made of molecules that can easily penetrate the tumor mass and many of these molecules can be supplied locally by cells nearby. C3b/iC3b deposited on tumor cells promotes adhesion of effector cells such as macrophages and NK cells through complement receptors, whereby cytotoxicity may ensue with the help of additional signals. A potential problem with this approach is the inhibitory effect of membrane-bound complement regulatory proteins (“mCRPs”) that are often overexpressed on tumor cells, which is a mechanism used by these cells evade complement attack (Jurianz et al., “Complement Resistance of Tumor Cells Basal and Induced Mechanisms,” Molecular Immunology 36:929-939 (1999)). However, efficient elimination of opsonized tumor cells can be achieved by blocking or overwhelming these mCRPs.
Compared to the protein-based reagents mentioned above, such as recombinant extracellular domains of complement receptors, monoclonal antibodies, and peptide aptamers, nucleic acid aptamers possess some compelling advantages. Nucleic acid molecules not only carry information in their linear sequences, but also fold into well-defined shapes that may be recognized specifically by proteins or other partners. The method known as in vitro selection or Systematic Evolution of Ligands by Exponential Enrichment (“SELEX”) attempts to generate novel nucleic acid ligands known as aptamers by applying genetic selection directly to a population of nucleic acid molecules through a process that emulates Darwinian evolution (Tuerk et al., “Systematic Evolution of Ligands by Exponential Enrichment: RNA Ligands to Bacteriophage T4 DNA Polymerase,” Science 249:505-510 (1990); Ellington et al., “In Vitro Selection of RNA Molecules that Bind Specific Ligands,” Nature 346:818-822 (1990)). To generate RNA aptamers, the SELEX experiment starts with a large randomized sequence pool containing 1014-1016 different species that fold into different shapes determined by their different sequences. This pool is then subjected to iterative cycles of selection and amplification. In each cycle, a target such as a protein molecule is used to select from the pool RNA molecules that bind it. Following the separation of bound RNA from unbound, the bound fraction is amplified by RT-PCR to generate a new pool for the next cycle. Usually, RNA ligands with the highest affinity for the target protein will dominate the population in 8-12 rounds. At the end of the process, the winning aptamers are cloned and sequenced for further characterization.
The aptamers generated by this process are capable of binding to a wide variety of targets with high affinity and specificity (Gold et al., “Diversity of Oligonucleotide Functions,” Annual Rev. Biochem. 64:763-797 (1995); Wilson et al., “In Vitro Selection of Functional Nucleic Acids,” Annual Rev. Biochem. 68:611-647 (1999)). Both DNA and RNA aptamers often bind their targets with dissociation constants (Kd) in the low nanomolar or picomolar range (1×10−7-10−12M) and are able to discriminate between related proteins that share common structural features. In addition to their widespread utility as molecular probes in basic research and diagnostic applications, aptamers are quickly becoming an exciting new class of therapeutic agents (Nimjee et al., “Aptamers: An Emerging Class of Therapeutics,” Annual Rev. Medicine 56:555-583 (2005)). With the advent of genomics and proteomics, protein-protein interaction is recognized as a promising category of drug targets. However, protein surfaces in direct contact with each other usually involve an area of about 1600 Å2 with relatively flat topography, causing concerns about the capacity for binding specificity of small molecules of less than 500 Da with less than 500 Å2 of total solvent-accessible surface area (Golemis et al., “Protein Interaction-targeted Drug Discovery: Evaluating Critical Issues,” BioTechniques 32:636-638, 640, 642 (2002); Lo Conte et al., “The Atomic Structure of Protein-protein Recognition Sites,” J. Mol. Biol. 285:2177-2198 (1999); Juliano et al., “Macromolecular Therapeutics: Emerging Strategies for Drug Discovery in the Postgenome Era,” Molecular Interventions 1:40-53 (2001)). Individual aptamers are usually 25-50 nucleotides long and weigh 8-16 kDa, which makes them better able to interact with proteins. Like antibodies, they can be made to order specifically for a particular protein. But unlike antibodies, aptamers are produced by a scalable in vitro process and display low to no immunogenicity or toxicity even when administered in pre-clinical doses 1000-fold greater than doses used in therapeutic application (Pendergrast et al., “Nucleic Acid Aptamers for Target Validation and Therapeutic Applications,” J. Biomolecular Techniques 16:224-234 (2005)).
Aptamers also compare favorably with other oligonucleotide-based pharmaceuticals. The targets of reagents like antisense and siRNA are located exclusively in the intracellular compartments as they act at the gene or mRNA level; delivery of these molecules to the target sites is a formidable task. In contrast, aptamers can exert their function against extracellular targets, which are much easier to access (Pestourie et al., “Aptamers Against Extracellular Targets for In Vivo Applications,” Biochimie 87:921-930 (2005)). Although natural RNA and DNA have poor pharmacokinetics when administered by intravenous or subcutaneous injection, they can be chemically improved to enhance their stability and to control their clearance (Pendergrast et al., “Nucleic Acid Aptamers for Target Validation and Therapeutic Applications,” J. Biomolecular Techniques 16:224-234 (2005)). For example, to render aptamers resistant to nuclease degradation, RNA modified at the 2′ position of pyrimidines with fluoro or amino groups can be used for selection. Additional post-selection modification or substitution can further increase aptamer residence time in the blood. For example, renal clearance of aptamers smaller than 40 kD can be reduced through conjugation with polyethylene glycol (“PEG”), attachment to liposomes, or cholesterol. Moreover, the activity of aptamers can be controlled by oligonucleotide antidotes that base-pair with the aptamers to prevent them from forming the correct shape to bind their targets (Rusconi et al., “Antidote-mediated Control of an Anticoagulant Aptamer In Vivo,” Nature Biotechnology 22:1423-1428 (2004)).
The first aptamer-based therapeutic approved for clinical use, Macugen (pegaptanib sodium injection), is a modified RNA aptamer directed to an isoform of vascular endothelial growth factor (VEGF165) and used to treat the wet form of age-related macular degeneration (“AMD”) as a locally acting drug (Ruckman et al., “2′-Fluoropyrimidine RNA-based Aptamers to the 165-amino Acid Form of Vascular Endothelial Growth Factor (VEGF165) Inhibition of Receptor Binding and VEGF-induced Vascular Permeability through Interactions Requiring the Exon 7-encoded Domain,” J. Biol. Chem. 273:20556-20567 (1998); Lee et al., “A Therapeutic Aptamer Inhibits Angiogenesis by Specifically Targeting the Heparin Binding Domain of VEGF165,” Proc. Nat'l Acad. Sci. U.S.A. 102:18902-18907 (2005)). The Macugen aptamer was generated by SELEX in the form of 2′ fluoro-pyrimidine RNA (2′F-Py RNA) and underwent post-selection 2′O-methyl-purine modifications. Its ends were further protected by 5′ PEG adducts and a 3′ dT attached via a 3′-3′ linkage. This aptamer binds to its target with a Kd of 50 pM and arrests the progression of wet AMD by preventing blood vessel growth. Clinical safety studies show that this drug is well tolerated at doses up to 10-fold higher than the 0.3-mg dose approved for the treatment of AMD (Chakravarthy et al., “Year 2 Efficacy Results of 2 Randomized Controlled Clinical Trials of Pegaptanib for Neovascular Age-related Macular Degeneration,” Ophthalmology 113:1508, e1501-1525 (2006); Apte et al., “Pegaptanib 1-year Systemic Safety Results from a Safety-pharmacokinetic Trial in Patients with Neovascular Age-related Macular Degeneration,” Ophthalmology 114:1702-1712 (2007)). In addition to Macugen, there are many other aptamers and aptamer-enabled technologies being evaluated in various stages of clinical trials for numerous diseases, including cancer. Notably, a 2′F-Py RNA aptamer for the prostate-specific membrane antigen (“PSMA”) was conjugated with docetaxel-encapsulated nanoparticles for targeted uptake by the prostate cancer cells (Lupold et al., “Identification and Characterization of Nuclease-stabilized RNA Molecules that Bind Human Prostate Cancer Cells Via the Prostate-specific Membrane Antigen,” Cancer Research 62:4029-4033 (2002); Farokhzad et al., “Targeted Nanoparticle-aptamer Bioconjugates for Cancer Chemotherapy in vivo,” Proc. Nat'l Acad. Sci. U.S.A. 103:6315-6320 (2006)). In a xenograft nude mouse model of prostate cancer, these bioconjugates showed significant anticancer efficacy without the systemic toxicity common to chemotherapeutics.
Because aptamers are produced by an in vitro process, the initial therapeutic leads can be isolated rapidly, and the production can be readily scaled up in a cost-effective manner. Because nucleic acids can easily regain activity following exposure to denaturing conditions, their shelf life is long. They can be administered by either intravenous or subcutaneous injection. Previously, aptamers against C5 have been developed as complement inhibitors (US Patent Application Publication No. 2006/0018871 A1) and aptamers against C3b have been similarly developed as complement inhibitors (PCT Application Publ. No. WO 97/42317).
The present invention is directed to individual aptamers for C3 and its derivatives as complement inhibitors, and composite aptamers binding C3 or its derivatives and a target protein to mediate opsonization by C3b/iC3b.